This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. A key step in determining the molecular architecture of cellular machinery using electron microscopy is being able to identify the individual molecules in 3-dimensional density maps. The molecules are tightly packed together making their boundaries hard to discern. Segmentation is the process of determining the regions of density maps for different molecules. We have developed a plug-in to UCSF Chimera called Segger that segments maps using automated computations based on the watershed algorithm, and allows hand-editing of the results to fix the inevitable mistakes guided by additional expert knowledge of the expected structures. The focus has been on electron microscopy of isolated molecular assemblies studied by single-particle electron cryo-microscopy, and there is strong interest in extending the software also be able to segment crowded cellular environments where a large number of diverse molecular assemblies are packed together.